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( 12 ) United States Patent US010208322B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 ,208 , 322 B2 Coelho et al. (45 ) Date of Patent: * Feb . 19, 2019 ( 54 ) IN VIVO AND IN VITRO OLEFIN ( 56 ) References Cited CYCLOPROPANATION CATALYZED BY HEME ENZYMES U . S . PATENT DOCUMENTS 3 , 965 ,204 A 6 / 1976 Lukas et al. (71 ) Applicant: California Institute of Technology , 4 , 243 ,819 A 1 / 1981 Henrick Pasadena , CA (US ) 5 ,296 , 595 A 3 / 1994 Doyle 5 , 703 , 246 A 12 / 1997 Aggarwal et al. 7 , 226 , 768 B2 6 / 2007 Farinas et al. ( 72 ) Inventors : Pedro S . Coelho , Los Angeles, CA 7 , 267 , 949 B2 9 / 2007 Richards et al . (US ) ; Eric M . Brustad , Durham , NC 7 ,625 ,642 B2 12 / 2009 Matsutani et al. (US ) ; Frances H . Arnold , La Canada , 7 ,662 , 969 B2 2 / 2010 Doyle et al. CA (US ) ; Zhan Wang , San Jose , CA 7 ,863 ,030 B2 1 / 2011 Arnold (US ) ; Jared C . Lewis , Chicago , IL 8 ,247 ,430 B2 8 / 2012 Yuan 8 , 993 , 262 B2 * 3 / 2015 Coelho . .. .. .. • * • C12P 7 /62 (US ) 435 / 119 9 ,399 , 762 B26 / 2016 Farwell et al . (73 ) Assignee : California Institute of Technology , 9 , 493 ,799 B2 * 11 /2016 Coelho .. C12P 7162 Pasadena , CA (US ) 2006 / 0030718 AL 2 / 2006 Zhang et al. 2006 / 0111347 A1 5 / 2006 Askew , Jr . et al. 2007 /0276013 AL 11 /2007 Ebbinghaus et al . ( * ) Notice : Subject to any disclaimer , the term of this 2009 /0238790 A2 9 /2009 Sommadosi et al. patent is extended or adjusted under 35 2010 / 0056806 A1 3 / 2010 Warren U . S . C . 154 (b ) by 0 days . 2010 /0168463 A1 7 /2010 Hirata et al. This patent is subject to a terminal dis 2010 /0240106 AL 9 /2010 Wong et al . claimer . 2011/ 0196086 A18 / 2011 Matsushita et al. ( Continued ) (21 ) Appl. No. : 15 / 278 , 561 FOREIGN PATENT DOCUMENTS (22 ) Filed : Sep . 28, 2016 EP 0 200 638 B1 4 / 1986 (65 ) Prior Publication Data WO 2007 / 144599 A2 12 / 2007 US 2017 / 0247725 A1 Aug. 31 , 2017 (Continued ) OTHER PUBLICATIONS Related U . S . Application Data Adams, P . et al ., “ Phenix : a comprehensive Python -based system for (63 ) Continuation of application No . 14 /625 , 449, filed on macromolecular structure solution ,” Acta Crystallogr ., Sect . D , Feb . 18 , 2015 , now Pat . No . 9 ,493 ,799 , which is a Biol. Crystallogr ., 2010 , D66 ( 2 ) : 213 -221 . continuation of application No . 14 / 185 , 861 , filed on ( Continued ) Feb . 20 , 2014 , now Pat . No . 8 , 993 , 262 , which is a continuation of application No . PCT /US2013 / 063577 , filed on Oct . 4 , 2013 . Primary Examiner — Pancham Bakshi (74 ) Attorney, Agent, or Firm — Kilpatrick Townsend & (60 ) Provisional application No . 61/ 711 ,640 , filed on Oct. Stockton LLP 9 , 2012 , provisional application No . 61 /740 , 247 , filed on Dec . 20 , 2012 , provisional application No . ABSTRACT 61 /784 ,917 , filed on Mar . 14 , 2013 , provisional (57 ) application No . 61/ 838 , 167 , filed on Jun . 21, 2013 , The present invention provides methods for catalyzing the provisional application No. 61/ 815 ,997 , filed on Apr . conversion of an olefin to any compound containing one or 25 , 2013 , provisional application No . 61/ 818 , 329 , more cyclopropane functional groups using heme enzymes . filed on May 1 , 2013 , provisional application No . In certain aspects , the present invention provides a method 61/ 856 ,493 , filed on Jul. 19 , 2013 . for producing a cyclopropanation product comprising pro (51 ) Int. CI. viding an olefinic substrate , a diazo reagent, and a heme C12P 13 / 02 ( 2006 .01 ) enzyme; and admixing the components in a reaction for a C12P 7762 ( 2006 . 01 ) time sufficient to produce a cyclopropanation product. In C12N 9 /02 ( 2006 . 01 ) other aspects , the present invention provides heme enzymes (52 ) U . S . CI. including variants and fragments thereof that are capable of CPC . .. .. C12P 13 /02 (2013 . 01 ) ; C12N 9 / 0004 carrying out in vivo and in vitro olefin cyclopropanation (2013 .01 ) ; C12N 9 /0042 (2013 . 01 ) ; C12P 7762 reactions . Expression vectors and host cells expressing the ( 2013 . 01 ) ; C12Y 106 / 02004 ( 2013 .01 ) ; YO2P heme enzymes are also provided by the present invention . 20 /52 (2015 . 11 ) (58 ) Field of Classification Search CPC .. .. C12P 13 /02 , C12P 7 /62 ; C12N 9 /0042 7 Claims, 46 Drawing Sheetsts See application file for complete search history . Specification includes a Sequence Listing . US 10 , 208 , 322 B2 Page 2 References Cited Davies , H . and Venkataramani, C . , “ Dirhodium tetraprolinate ( 56 ) catalyzed asymmetric cyclopropanations with high turnover num U . S . PATENT DOCUMENTS bers , ” Org . Lett ., 2003 , 5 ( 9 ) : 1403 - 1406 . Davies , H . and Manning , J . , “ Catalytic C - H functionalization by 2012 /0237591 A1 9 / 2012 Cullis et al . 2013 /0165696 A1 6 / 2013 Khile et al. metal carbenoid and nitrenoid insertion , ” Nature, 2008 , 451 (7177 ) :417 2014 / 0242647 AL 8 / 2014 Coelho et al. 424 . 2015 / 0232814 Al 8 / 2015 Farwell et al. Davies , H . and Beckwith , R ., “ Catalytic enantioselective C - H 2015 /0267232 A1 9 / 2015 Coelho et al . activation by means of metal- carbenoid - induced C - H insertion , " 2016 / 0002682 AL 1 / 2016 Coelho et al. Chem . Rev . , 2003 , 103 ( 8 ) : 2861- 2903 . 2016 / 0032330 A1 2 / 2016 Renata et al . Dawson , J . , “ Probing structure- function relations in heme 2016 / 0040199 Al 2 / 2016 Hyster containing oxygenases and peroxidases ,” Science , 1988 , 240 (4851 ) :433 9 . FOREIGN PATENT DOCUMENTS Donaldson , W . , “ Synthesis of cyclopropane containing natural WO 2011 / 159550 A3 12/ 2011 products, ” Tetrahedron , 2001, 57 (41 ) :8589 - 8627 . WO 2013 / 124280 AL 8 / 2013 Doyle , M . et al. , “ Dirhodium ( II ) tetrakis [methyl 2 - oxaazetidine - 4 WO 2013 / 151690 A1 10 / 2013 carboxylate ] : a chiral dirhodium ( II ) carboxamidate of exceptional WO 2014 /058729 A14 / 2014 reactivity and selectivity , " Org . Lett ., 2000 , 2 ( 8 ) : 1145 - 7 . WO 2014 /058744 A2 4 / 2014 Dunford , A . et al ., “ Probing the molecular determinants of coen WO 2016 /086015 A1 6 / 2016 zyme selectivity in the P450 BM3 FADNADPH domain , " Biochimica WO 2016 / 191612 A2 12 / 2016 Biophysica Acta , 2009 , 1794 ( 8 ) : 1181 - 1189 . Emsley, P . and Cowtan , K ., “ Coot : model -building tools for molecu lar graphics, ” Acta Crystallogr ., Sect. D , Biol. Crystallogr. , 2004 , OTHER PUBLICATIONS D60 ( 12 , Pt. 1 ) :2126 -2132 . Ajikumar , P . et al ., “ Isoprenoid pathway optimization for taxol Evans, D . et al. , “ Bis ( oxazolines) as chiral ligands in metal catalyzed asymmetric reactions . Catalytic , asymmetric cyclopropana precursor overproduction in Escherichia coli, ” Science, 2010 , 330 : 70 tion of olefins , " J . Am . Chem . Soc . , 1991, 113 ( 2 ) : 726 - 8 . 74 . Evans, P . , “ Scaling and assessment of data quality, ” Acta Crystal Alliot, J. et al. , “ Enantioselective synthesis of levomilnacipran ," logr . , Sect . D , Biol. Crystallogr. , 2006 , D62( 1 ) :72 - 82 . Chem . Commun . , 2012 , 48 (65 ) :8111 - 8113 . Galardon , E . et al. , “ Insertion of ethyl diazoacetate into N - H and Altschul, S . et al. , “ Basic local alignment search tool, ” J . Mol. Biol ., S - H bonds catalyzed by ruthenium porphyrin complexes, ” J . Chem . 1990 , 215 ( 3 ) :403 - 10 . Perkin Tans, 1 : 2455 - 2456 , 1997 . Bailey, S . , “ The CCP4 suite : programsfor protein crystallography, " Girvan et al. , " Glutamate -Heme Ester Bond Formation Is Disfavoured Acta Cryst . , Sect. D Biol. Crystallogr ., 1994 , D50 ( 5 ) :760 - 763. in Flavocytochrome P450 BM3, ” Biochemical Journal, 2010 , pp . Baumann , L . et al. , “ Iron porphyrin catalyzed N - H insertion reac 455 -466 . tions with ethyl diazoacetate , " Organometallics , 2007 , 26 :3995 Green , M . , “ C - H bond activation in heme proteins : the role of 4002 . thiolate ligation in cytochrome P 450 , ” Curr . Opin . Chem . Biol . , Bergman , R ., “ Organometallic chemistry : C - H activation , ” Nature , 2009 , 13 ( 1 ) : 84 - 88 . 2007 , 446 (7134 ) :391 - 393 . Groves , J . , “ The bioinorganic chemistry of iron in oxygenases and Bloom , J. et al. , " Protein stability promotes evolvability , ” Proc . supramolecular assemblies , ” Proc. Natl . Acad . Sci . U . S . A . , 2003 , 100 ( 7 ) : 3569 - 3574 . Natl. Acad . Sci. U . S . A ., 2006 , 103 ( 15 ) : 5869 - 5874 . Haines , D . et al. , “ Pivotal Role of Water in the Mechanism of Bonnaud , B . et al. , “ 1 - Aryl - 2 - aminomethyl) cyclopropanecarboxylic P450BM - 3 , ” Biochemistry , 2001 , 40 ( 45 ) : 13456 - 13465 . acid derivatives. A new series of potential antidepressants, " J . Med . Heel et al . , “ Non - natural Olefin Cyclopropanation Catalyzed by Chem ., 1987 , 30 ( 2 ) : 318 - 25 . Diverse Cytochrome P450s and Other Hemoproteins” , ChemBioChem Bornscheuer , U . and Kazlauskas , R ., “ Reaction specificity of enzymes : vol. 15 , Iss . 17 , Oct . 7 , 2014 , pp . 2556 - 2562 . Catalytic promiscuity in biocatalysis : Using old enzymes to form Hiraga , K . and Arnold , F ., “General method for sequence new bonds and follow new pathways, ” Angew . Chem . , Int. Ed . , independent site - directed chimeragenesis , " J . Mol . Biol. , 2003 , 2004 , 43 (45 ) :6032 -6040 . 330 ( 2 ) : 287 - 96 . Boyce , M . and Bertozzi , C ., “ Bringing chemistry to life ,” Nat . Hüttinger , K . , “ Semi- synthetic proteins for catalytic and analytical Methods , 2011, 8 ( 8 ) :638 -642 . applications, ” May 2009 , Georgia Institute of Technology . Breslow , R ., “ Biomimetic chemistry : Biology as an inspiration ,” J . Hyster, T . et al ., “ Biotinylated Rh ( III ) complexes in engineered Biol. Chem . , 2009 , 284 ( 3 ) : 1337
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